Microglial cells are involved in the susceptibility of NADPH oxidase knockout mice to 6-hydroxy-dopamine-induced neurodegeneration.

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.

Abstract

We explored the impact of Nox-2 in modulating inflammatory-mediated microglial responses in the 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD) model. Nox1 and Nox2 gene expression were found to increase in striatum, whereas a marked increase of Nox2 expression was observed in substantia nigra (SN) of wild-type (wt) mice after PD induction. Gp91(phox-/-) 6-OHDA-lesioned mice exhibited a significant reduction in the apomorphine-induced rotational behavior, when compared to wt mice. Immunolabeling assays indicated that striatal 6-OHDA injections reduced the number of dopaminergic (DA) neurons in the SN of wt mice. In gp91(phox-/-) 6-OHDA-lesioned mice the DA degeneration was negligible, suggesting an involvement of Nox in 6-OHDA-mediated SN degeneration. Gp91(phox-/-) 6-OHDA-lesioned mice treated with minocycline, a tetracycline derivative that exerts multiple anti-inflammatory effects, including microglial inhibition, exhibited increased apomorphine-induced rotational behavior and degeneration of DA neurons after 6-OHDA injections. The same treatment also increased TNF-α release and potentiated NF-κB activation in the SN of gp91(phox-/-)-lesioned mice. Our results demonstrate for the first time that inhibition of microglial cells increases the susceptibility of gp91(phox-/-) 6-OHDA lesioned mice to develop PD. Blockade of microglia leads to NF-κB activation and TNF-α release into the SN of gp91(phox-/-) 6-OHDA lesioned mice, a likely mechanism whereby gp91(phox-/-) 6-OHDA lesioned mice may be more susceptible to develop PD after microglial cell inhibition. Nox2 adds an essential level of regulation to signaling pathways underlying the inflammatory response after PD induction.

Effect of 6-OHDA on mRNAs of Nox1 (A), Nox2 (B) and Nox4 (C) isoforms in the striatum. HPRT was used as an internal control; n=6. The statistical significance is expressed as: * P < 0.05. In D and I, representative Western blots illustrating p67phox protein content in cytosolic and membrane fractions obtained from striatum and SN respectively, after 6-OHDA-induced PD. Six-OHDA did not induce significant p67phox translocation from the cytosol to the plasma membrane in none of the analyzed samples; n=4 tested samples. In E and J, effect of 6-OHDA on striatal and SN Rac-1 total protein levels. The graphs represent the mean ratio of Rac-1 densitometric data in relation to β-actin of 5 tested samples. Significance compared to Wt saline at **p <0.01. In F, effect of 6-OHDA on mRNAs of Nox1 (F), Nox2 (G) and Nox4 (H) isoforms in the SN. HPRT was used as an internal control; n=6. The statistical significance is expressed as: * P < 0.05.

Striatal concentration of pro-inflammatory (IL-1β, TNF-α, IFN-γ, IL-2) and anti-inflammatory cytokines (IL-10 and TGF-β1) and the chemokine RANTES following 6-OHDA-induced PD in Wt and gp91phox-/- treated with minocycline or PBS. All concentrations were expressed in pg/ml. The statistical significance is expressed as: * P<0.05; ** P<0.01 and *** P<0.001; n=6 for each experimental group tested.

Concentration of pro-inflammatory (IL-1β, TNF-α, IFN-γ, IL-2) and anti-inflammatory cytokines (IL-10 and TGF-β1) and the chemokine RANTES in the substantia nigra following 6-OHDA-induced PD in Wt and gp91phox-/- treated with minocycline or PBS. All concentrations were expressed in pg/ml. The statistical significance is expressed as: * P<0.05; ** P<0.01 and *** P<0.001; n=6 for each experimental group tested.